Cobabalamin is an essential nutrient to most organisms, including humans. Structurally, cobalamin is an elaborate molecule composed of a cobalt-containing macrocyclic tetrapyrrole, the corrin ring, a nucleotide loop whose base serves as a lower ligand to the cobalt, and an amino-propanol moiety that joins the nucleotide loop to the corrin macrocycle. The structure of cobalamin is shown in FIG. 1. Cobalamin molecules may contain different chemical groups as upper ligands to the cobalt in the corrin ring (e.g. xe2x80x94OH, xe2x80x94H2O, xe2x80x94CN, xe2x80x94CH3 and adenosyl).
Cyanocobalamin, also known as vitamin B12, is a form of cobalamin that contains a cyano (CN) group as an upper ligand. This is the form of cobalamin that is made and sold commercially and is also available as a chemical reagent on the commercial market. The biologically active form of cobalamin contains an adenosyl (Ado) group as the upper ligand to the cobalt. Non-biologically active forms of cobalamin, such as vitamin B 12, must therefore be converted to adenosylcobalamin for use by living organisms. Conversion from cyanocobalamin to adenosylcobalamin requires the reduction of the cobalt atom from Co[III] to the Co[I] oxidation state as well as the transfer of the adenosyl moiety of ATP to the Co[I] form of cobalamin. The enzyme that catalyzes the transfer of the adenosyl group to the corrin ring, an ATP:corrinoid adenosyltransferase (CobA), has been identified and characterized in several organisms in which this process has been studied. In order for adenosyltransferase to function, the cobalt atom must be in the Co[I] oxidation state, but this enzyme is not capable of reducing the oxidation state of the cobalt molecule in cobalamin.
Biological production of chemicals with industrial value has been greatly improved by the use of large scale bacterial cultures employing genetically engineered strains. The efficient operation of several of such bacterial production processes requires the addition of exogenous adenosylcobalamin to the culture medium. While adenosylcobalamin can be added to such a culture, it is a relatively expensive constituent that can raise the cost of the overall process. In addition, the inability of the bacterial culture to recycle adenosylcobalamin can lead to significantly lower yields of the desired product. This raises the need for a system in which bacterial hosts can be engineered to generate and recycle adenosylcobalamin to make the overall production process more cost-effective and increasing yields.
It is first disclosed here that a combination of the enzymes ATP:corrinoid adenosyltransferase (CobA) and flavodoxin (FldA) are both necessary and sufficient for a bacteria in fermentation culture to produce adenosylcobalamin from cyanocobalamin. By also adding the enzyme flavodoxin (ferrodoxin):NADP+ reductase (Fpr), the FldA can be recycled so that the adenosylcobalamin can be recycled continuously during a fermentation process.
It is also taught here that bacterial expression vectors can be constructed containing genes encoding the expression of both CobA and FldA in bacterial hosts. The transformation of such expression vectors into a fermentation host confers upon that host the ability to produce adenosylcobalamin from cyanocobalamin, thereby lowering the fermentation costs for that bacteria. The expression vector can also carry a construct encoding the expression of Fpr.
It is an advantageous variant of the present invention that genes encoding all three enzymes, CobA, FldA and Fpr, can be combined on a single plasmid to make a plasmid that, when transferred into a bacterial host, makes that host independent of the need for external adenosylcobalamin in fermentation culture.
Other objects advantages and features of the present specification will become apparent from the following specification.